Epigenetic reprogramming, the systematic removal of molecular markers on DNA and histones, occurs during early mammalian embryogenesis and is thought to be crucial for normal development. The mechanism by which specific chromosomal regions are targeted for reprogramming is not fully understood. Recent research in mammalian cell lines has indicated that siRNAs homologous to promoter regions of specific genes initiate transcriptional gene silencing, suggesting that the RNAi machinery is involved in chromatin silencing in mammalian cells. However, a biological role for mammalian RNAi in transcriptional silencing has not been established. We propose to determine if small RNA mediated chromatin silencing is occurring in ES cells. We will assess the effects of Argonaute protein silencing on chromatin structure in ES cells before and after differentiation by evaluating changes in DNA methylation and histone modification. The mechanism by which the Argonuate proteins function and the role of small RNA molecules in chromatin silencing will be addressed. These studies will provide new insights into the role of small RNA molecules and the Argonaute protein family in hetrochromatin formation during development.